ᮞᩩ࠽ࡻࡦムⷾ

(1) Hybond P⭯ (Amershamacia) (2) Blotting buffer

80 % 1™Running bufferࠉ20 % MeOH (3) Blocking buffer

1.5 gࡡBSAࠉ࠵ࡾ࠷ࡢ skim milkࢅ30 mLࡡ0.05 % tween-PBS࡞⁈ゆࡊࠉ150 ȣLࡡ20 % NaN3ࢅຊ࠻࡙౐⏕ࡊࡒࠊ

(4) ୌḗᢘమᕵ㔐ᾦ

0. 05% tween-PBSࠉ偏偃BSAࠉ0.1%NaN3

(5) ஦ḗᢘమཬᚺᾦ

10 mLࡡ0.05% tween-PBS࡞0.30 gࡡBSAࢅ⁈ゆࡊࠉ5ȣLࡡ஦ḗᢘమࢅຊ࠻

࡙౐⏕ࡊࡒࠊ 13.2.2 ᐁ㥺᪁Ἢ

SDS-PAGE ࡞࡙ฦ㞫ࡊࡒࢰࣤࣂࢠ㈹ࢅࢗ࢘ࢴࢹ᪁ᘟࣇࣞࢴࢹ⿞⨠ࢅ⏕࠷࡙

Hybond P⭯࡞30 Vࡡᏽ㞹ᅸ࡚4 hr࠾ࡼୌᬄࢹࣚࣤࢪࣆ࢒࣭ࡊࡒࠊ⩛᪝ࠉmembrane

ࢅBlocking buffer࡞ᾈࡊᐄῺ࡚60 min㟀┖ࡊࡒࠊTween-PBS࡚ὑὯࡊ5 min㟀

┖ࡈࡎࡾࡆ࡛ࢅ 3 ᅂ⧖ࡽ㏁ࡊࡒᚃࠉୌḗᢘమࢅཬᚺࡈࡎࡒࠊ⇆ᵕ࡞ Tween-PBS

࡚ᩐᅂὑὯࠉ㟀┖ࢅ⧖ࡽ㏁ࡊࠉPBS࡚ὑὯᚃࠉ஦ḗᢘమࢅᐄῺ࡚60 minཬᚺࡈ ࡎࡒࠊ්ᗐ Tween-PBS ࡚3 ᅂὑὯࠉ㟀┖ࢅ⧖ࡽ㏁ࡊࠉPBS࡚ 3 ᅂὑὯࠉ5 min 㟀┖ࡈࡎࡒࠊⓆⰅ࡞ࡢECL^TMKitࢅ⏕࠷࡙ࣆ࢔࣑ࣜ࡞᳠ฝࡊࡒࠊ

13.2.3 ᳠ฝ࡞⏕࠷ࡒᢘమ

primary Ab

isotype dilution reaction

Șlamin B1 mouse IgG1ȡ ™1000 r.t. for 1 hr

Ș phosphorylated Chk1 (Ser345)

rabbt IgG ™1000 4Υ for O/N

Ș LC-3 rabbt IgG ™2000 r.t. for 1 hr

Ș HA rat IgG ™3000 r.t. for 1 hr

ȘȘ-tubulin mouse IgG₁ ™10000 r.t. for 1 hr

Ș ș-actin mouse IgG ™1000 r.t. for 1 hr

Secondary Ab

dilution reaction

HRP conjugated Ș mouse IgG

™2000 r.t. for 1 hr

HRP conjugated Ș rabbit IgG

™3000 r.t. for 1 hr

ޥ辞

ᮇ◂✪࡞࠵ࡒࡽࠉᚒᣞᑙᚒ㠬᧙ࢅ㈯ࡽࡱࡊࡒ᮶ℓኬᏕኬᏕ㝌ⷾᏕ◂✪⛁㐿ఎᏄⷾᏕฦ㔕ᩅ᤭ ᴦ ᮇに⨶ ඙⏍࡞ㅵࢆ࡚ᚒ♡⏞ࡊ୕ࡅࡱࡌࠊ

ᮇㄵᩝࢅࡱ࡛ࡴࡾ࡞࠵ࡒࡽࠉ᭯─࡝ࡾᚒຐゕࢅ㡤ࡀࡱࡊࡒ᮶ℓኬᏕኬᏕ㝌ⷾᏕ◂✪⛁⏍మ㜭ᚒⷾ

Ꮥฦ㔕෶ᩅ᤭ ஁ୖ࿔ఫ ඙⏍࡞῕ࡂវㅨ⮬ࡊࡱࡌࠊ

ᮇ◂✪ࢅ㐅ࡴࡾ࡞࠵ࡒࡽࠉ⤂╂᭯─࡝ᚒᣞᑙᚒຐゕࢅ㡤ࡀࡱࡊࡒ᮶ℓኬᏕኬᏕ㝌ⷾᏕ◂✪⛁㐿ఎ ᏄⷾᏕฦ㔕෶ᩅ᤭ 㛭ᨳᖶ ඙⏍࡞῕ࡂᚒ♡⏞ࡊ୕ࡅࡱࡌࠊ

ᮇ◂✪ࢅ㐅ࡴࡾ࡞࠵ࡒࡽࠉኣኬ࡝ࡾᚒຐゕᚒᣞᑙࢅ㡤ࡀࡱࡊࡒ᮶ℓኬᏕኬᏕ㝌ⷾᏕ◂✪⛁㐿ఎᏄ

ⷾᏕฦ㔕ຐᡥ ྚᮟ᪺ ඙⏍࡞῕ࡂᚨࡻࡽវㅨ⮬ࡊࡱࡌࠊ

ᮇ◂✪ࢅ㐅ࡴࡾ࡞࠵ࡒࡽࠉ├᥃ᚒᣞᑙ࠷ࡒࡓࡀࡱࡊࡒࠉ᮶ℓኬᏕኬᏕ㝌ⷾᏕ◂✪⛁㐿ఎᏄⷾᏕฦ 㔕COEᢇ⾙⿭ఫဤ ஬୕⤦㔓ᵕ࡞ᚨࡻࡽᚒ♡⏞ࡊ୕ࡅࡱࡌࠊ

ᮇ◂✪ࢅ㐅ࡴࡾ࡞࠵ࡒࡽࠉᚒᣞᑙᚒຐゕࢅ㡤ࡀࡱࡊࡒ᮶ℓኬᏕኬᏕ㝌ⷾᏕ◂✪⛁㐿ఎᏄⷾᏕฦ㔕

ຐᩅ ኣ⏛࿔ྎ ඙⏍࡞῕ࡂᚒ♡⏞ࡊ୕ࡅࡱࡌࠊ

᭩ᚃ࡞࡝ࡽࡱࡊࡒ࠿ࠉ◂✪⏍Ὡࢅ㏞ࡾ࡞࠵ࡒࡽࠉᖏ࡞ᨥ࠻࡙㡤ࡀࡱࡊࡒ⣵⬂ࢲ࣭࣑ࡡⓑᵕࠉ⇆᭿ ࡡ᪁ࠍࢅ╂ࡴ࡛ࡌࡾ㐿ఎᏄⷾᏕฦ㔕ࡡ᪁ࠍ࡞ᚨࡻࡽᚒ♡⏞ࡊ୕ࡅࡾ࡛࡛ࡵ࡞ࠉ௑ᚃࡡᚒὩ㌅ࢅ࠽

♫ࡽࡊࡱࡌࠊ 㧏⏛ 㞑ஒ

ᘤ⏕ᩝ⊡

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